Data for modelling included known prognostic factors MGMT status, age, corticosteroid use and ECOG performance status, and multifocal disease. MGMT promoter methylation status was determined by using pyrosequencing on purified DNA with the Therascreen MGMT Pyro Kit (Qiagen). This analysis was performed on all diagnostic samples, including stereotactic biopsies, and mean methylation above 10% was considered positive. More details of the biomarker analysis are described in a previous paper by Abedi et al.5 (link). The Eastern Cooperative Oncology Group (ECOG) performance status scale was used to assess the functional status of each patient, ranging from 0 for fully active to 5 for diseased, and was registered at the start of concomitant treatment26 (link). Patients receiving at least 10 mg/day of prednisolone when beginning concomitant treatment were corticosteroid users.
Therascreen mgmt pyro kit
Manufactured by Qiagen
Sourced in Germany
The Therascreen MGMT Pyro Kit is a laboratory instrument designed for the detection and quantification of methylation patterns in the MGMT gene promoter region. It utilizes pyrosequencing technology to analyze DNA samples and provide information about the methylation status of the MGMT gene.
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Lab products found in correlation
Epitect bisulfite kit, by Qiagen (5 mentions)
Pyromark q24 system, by Qiagen (5 mentions)
Epitect fast dna bisulfite kit, by Qiagen (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Pyromark q24, by Qiagen (3 mentions)
Vs120 slide scanner, by Olympus (2 mentions)
Clone 3c6, by Roche (1 mentions)
Pyromark pcr kit 200, by Qiagen (1 mentions)
Ffpe tissue kit, by Qiagen (1 mentions)
Qiamp dna mini kit, by Qiagen (1 mentions)
Qiacube automated purification system, by Qiagen (1 mentions)
Pyromarktm q24, by Qiagen (1 mentions)
Hotstartaq dna polymerase, by Qiagen (1 mentions)
Methylationepic 850k array platform, by Illumina (1 mentions)
Mytaq polymerase, by Meridian Bioscience (1 mentions)
Bigdye terminator v3, by Thermo Fisher Scientific (1 mentions)
Abi 3500 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna ffpe tissue kit, by Qiagen (1 mentions)
19 protocols using therascreen mgmt pyro kit
1
Prognostic Factors in Glioblastoma
2
Glioma Tissue Characterization by Multimodal Imaging
3
Molecular Profiling of IDH1 Wild-Type GBM
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Neuropathological diagnosis including molecular markers of the tumor was routinely assessed using standard histopathology, immunohistochemistry, in situ hybridization, and sequencing technologies. For molecular classification, following markers, among others, were determined in routine analysis: IDH1 status, MGMT methylation status and EGFR amplification status. The MGMT methylation status was assessed by pyrosequencing using the Therascreen MGMT Pyro Kit (QIAGEN, Hilden, Germany). Cases with a mean methylation percentage of more than 8% were considered to be MGMT methylated [20 (link)]. Immunohistochemistry was performed to assess EGFR amplification status (clone 3C6; Ventana, Oro Valley, AZ, USA) and IDH1 mutation status (clone DIA-H09; Dianova, Hamburg, Germany) [21 (link),22 (link)]. Presence of oligodendroglial component was excluded by 1p19q codeletion status.
Because of clinical and prognostic impact, in this study we focused only on MGMT and EGFR alterations in IDH1 wild type GBM.
Because of clinical and prognostic impact, in this study we focused only on MGMT and EGFR alterations in IDH1 wild type GBM.
4
Methylation Analysis of MLH1 and MGMT Genes
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In order to determine the percentage of methylation of the samples studied in the MLH1 and MGMT genes, pyrosequencing analysis was conducted. Samples were initially embedded in paraffin. The total DNA was extracted and treated with bisulphite, using the Epitect Fast DNA Bisulfite Kit (Qiagen®, Hilden, Germany), and following the protocol recommended by the manufacturer: one 5 μm cut of paraffin for MHL1 and two 5 μm cuts for MGMT. The concentration of the samples was measured using the NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA), and all of the samples had 260/280 values of between 1.7–2. Once transformed, the DNA was extracted, and the Therascreen MGMT Pyro kit (Qiagen®) was then used to perform PCR and pyrosequencing for methylated MGMT detection. With regards to MLH1, the PCR was performed using the Pyromark PCR kit 200 (Qiagen®) with a concentration of no greater than 100 ng/microliter. The pyrosequencing was conducted using Pyromark Q24 CPG MLH1 (4 × 24) (Qiagen®). All PCRs were performed in the Agilent Technologies Surecycler 8800, and the pyrosequencing was performed by the Pyromark Q24 and Pyromark Q24 workstation (Qiagen®), using the Pyromark Q24 2.0.7 to compute and analyze the results.
5
MGMT Methylation Assessment Protocol
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In the initial clinical trial protocol that was developed in the year 2009, MGMT testing was not established as standard procedure for patients included in the trial; it was added ex post. A retrospective collection of samples for MGMT analysis was thus only successful in a fraction of patients (see Section 2 . Results). Depending on availability, DNA was extracted from either tumor tissue or formalin-fixed paraffin-embedded tissue (FFPE) sections using the QiAmp DNA Mini-Kit and FFPE tissue kit (both Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Subsequently, DNA modification was performed with the EpiTect Bisulfite Kit (Qiagen). Using the Therascreen MGMT Pyro Kit (Qiagen), bisulfite converted genomic DNA was amplified by PCR and sequenced on a Pyromark Q24 (Qiagen, Germany) system. Following previous research [58 (link)], a mean value of the methylation percentage of the four CpG sites investigated was calculated. Accordingly, a mean methylation >8% was considered as methylated.
6
MGMT Promoter Methylation Analysis
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The MGMT analysis was done on the DNA extracted from paraffin-embedded tumor samples after bisulfite treatment and PCR amplification with primers specific for exon 1 of MGMT. Preliminarily, unmethylated cytosine residues were converted to uracil with bisulfite treatment of 500 ng DNA using the Epi Tect Bisulfite Kit (Qiagen) and the QiaCube automated purification system (Qiagen) according to the manufacturer's recommendation. The Therascreen MGMT Pyro Kit and the PyroMark Q24 system (Qiagen) were used to assess the methylation status of the MGMT gene promoter. Briefly, bisulfite-converted genomic DNA was amplified by PCR, the amplicons were immobilized on streptavidin beads, and single-stranded DNA was prepared, sequenced, and finally analyzed on the PyroMark Q24 system. The cut-off frequency for accepting methylation as positive was determined as elsewhere reported (46 (link)).
7
MGMT Promoter Methylation Analysis
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To determine the MGMT promoter methylation status, genomic DNA from U251-MG cell lines (see 4.6 for isolation protocol) were analysed in the Department of Neuropathology, at the University Hospital Erlangen, Germany. According to manufacturer’s guidelines, samples were firstly bisulfide converted by using the EpiTect Bisulfite Kit (Qiagen, Hilden, Germany) and the prescribed sequence of interest was afterwards sequenced via pyro-sequencing with the therascreen MGMT Pyro Kit (Qiagen).
Sequence used for analysing:
Sequence used for analysing:
MGMT: 5′-C/TGTTTTGC/TGTTTC/TGAC/TGTTC/TGTAGGTTTTC/TGC/TG-3′.
8
Promoter methylation analysis protocol
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The assays used in this study targeted promoters of RUNX3, ABCB1, TRIM58, and TNF1 genes and were previously published. The details of the assay designs are shown in Table 1 . We also used one commercially available assay, therascreen® MGMT Pyro® Kit (Qiagen Gmbh, Hilden, Germany), targeting exon 1 of MGMT gene.
The pre pyrosequencing PCRs contained 10× PCR Buffer, 1.6 mM MgCl2, dNTP mix (0.4 mM of each), 1U of HotStarTaq DNA Polymerase (QIAGEN GmbH, Hilden, Germany), 200 nM of each primer, and 200–500 ng input DNA. The PCR amplifications were performed as in the original publications (Table 1 ) for RUNX3, ABCB1, TRIM58, and TNF1 genes and as recommended by the manufacturer for the MGMT gene. Pyrosequencing of the amplified PCR product was carried out using the PyroMarkTM Q24 instrument (QIAGEN GmbH, Hilden, Germany) according to the manufacturer protocol. All the experiments were repeated at least three times for each of the analysed sequences.
The pre pyrosequencing PCRs contained 10× PCR Buffer, 1.6 mM MgCl2, dNTP mix (0.4 mM of each), 1U of HotStarTaq DNA Polymerase (QIAGEN GmbH, Hilden, Germany), 200 nM of each primer, and 200–500 ng input DNA. The PCR amplifications were performed as in the original publications (
9
MGMT Methylation Assessment in Glioma
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All patients underwent either biopsy or resection following a baseline MRI. The tissue was sent to neuropathology for further diagnostics. The MGMT methylation status was assessed by pyrosequencing, using the Therascreen MGMT Pyro Kit (QIAGEN, Hilden, Germany). Tumors with a mean methylation percentage of more than 8% were considered to be MGMT methylated [30 (link)].
10
Methylation-based Glioma Profiling
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Tumor tissues were selected based on their methylation data from the database of the Department of Neuropathology Heidelberg62 (link). All methylation data were generated using the Illumina MethylationEPIC (850k) array platform according to the manufacturer’s instructions (Illumina, San Diego, USA). Sample preparation was performed as previously described63 (link). DNA methylation status of 10.000 CpG sites was analyzed on the current version v12b4 of the Classifier (https://www.molecularneuropathology.org/mnp ). Of note, all cases presented a calibrated Classifier Score > 0.9.
The probability of MGMT promoter methylation from 850k array data was estimated as previously described64 (link). For cases with an MGMT promoter methylation status not determinable by 850k methylation analysis, additional pyrosequencing was performed using the therascreen® MGMT Pyro® kit (QIAGEN®) and the PyroMark® Q24 system (QIAGEN®) according to the manufacturer’s protocol. Bisulfite conversion was done with the EpiTect fast DNA bisulfite kit (QIAGEN®). In accordance with published studies65 (link)–67 (link), a mean MGMT promoter methylation percentage < 8% across the investigated CpG sites was considered as non-methylated and a value ≥8% was considered as methylated.
The probability of MGMT promoter methylation from 850k array data was estimated as previously described64 (link). For cases with an MGMT promoter methylation status not determinable by 850k methylation analysis, additional pyrosequencing was performed using the therascreen® MGMT Pyro® kit (QIAGEN®) and the PyroMark® Q24 system (QIAGEN®) according to the manufacturer’s protocol. Bisulfite conversion was done with the EpiTect fast DNA bisulfite kit (QIAGEN®). In accordance with published studies65 (link)–67 (link), a mean MGMT promoter methylation percentage < 8% across the investigated CpG sites was considered as non-methylated and a value ≥8% was considered as methylated.
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